Stenotrophomonas maltophilia is an important multidrug-resistant nosocomial pathogen associated with high mortality. Our aim was to examine antimicrobial susceptibility, biofilm production and clonal relatedness of clinical isolates of S. maltophilia. S. maltophilia isolates were collected between 2006 and 2013 from two tertiary care hospitals in Mexico. Antimicrobial susceptibility was evaluated by the broth microdilution method. PCR was used to determine the presence of b-lactamase genes L1 and L2. Biofilm formation was assessed with crystal violet staining. Clonal relatedness was determined by PFGE. Among the 119 collected S. maltophilia isolates, 73 (61.3 %) were from the respiratory tract. Resistance levels exceeded 75 % for imipenem, meropenem, ampicillin, aztreonam, gentamicin and tobramycin. Resistance to trimethoprim-sulfamethoxazole was 32.8 %. L1 and L2 genes were detected in 77.1 % (91/118) and 66.9 % (79/118) of isolates, respectively. All S. maltophilia strains were able to produce biofilms. Strains were classified as weak (47.9 %, 57/119), moderate (38.7 %, 46/119), or strong (13.4 %, 16/119) biofilm producers. A total of 89 distinct PFGE types were identified and 21.6 % (22/102) of the isolates were distributed in nine clusters. This is the first study in Mexico to reveal characteristics of clinical isolates of S. maltophilia. Clonal diversity data indicate low crosstransmission of S. maltophilia in a hospital setting. The high antibiotic resistance underscores the need for continuous surveillance of S. maltophilia in hospital settings in Mexico.

Non-typeable Haemophilus influenzae (NTHi) is a common opportunistic bacterial pathogen that primarily infects the respiratory mucosa. This study was conducted to assess clinical and microbiological data related to disease severity in patients with lower respiratory tract infections caused by NTHi in a tertiary care hospital in Mexico. NTHi isolates were subjected to serotyping, antimicrobial susceptibility evaluationand analyses of β-lactamase production, genetic relatednessand biofilm formation. Clinical and demographic data were retrieved from patients' records. The mean age of the patients was 40.3 years; the majority (n=44, 72.1 %) were male. The main comorbidities were arterial hypertension (n=22, 36.1 %) and diabetes mellitus (n=17, 27.9 %). NTHi isolates (n=98) were recovered from tracheal aspirate (n=57, 58.2 %), sputum (n=26, 26.5 %)and bronchial aspirate (n=15, 15.3 %) specimens. Low resistance to cefotaxime (n=0, 0.0 %), rifampin (n=1, 1.1 %) and chloramphenicol (n=3, 3.2 %) and greater resistance to ampicillin (n=30, 32.3 %) and trimethoprim-sulfamethoxazole (n=49, 52.7 %) were detected. β-Lactamase production was found in 17 (17.3 %) isolates. Isolates displayed high genetic diversity, and only 10 (10.2 %) were found to be biofilm producers. The antimicrobial susceptibility patterns of biofilm-producing and non-producing isolates did not differ. Biofilm production was associated with prolonged hospital stay (P=0.05). Lower respiratory NTHi isolates from Mexico showed low antimicrobial resistance and weak biofilm production. Younger age was correlated with lower Acute Physiology and Chronic Health Evaluation II score (moderate, P=0.07; severe, P=0.03).

Infections and disease caused by the obligate human pathogen Bordetella pertussis (Bp) are increasing, despite widespread vaccinations. The current acellular pertussis vaccines remain ineffective against nasopharyngeal colonization, carriage, and transmission. In this work, we tested the hypothesis that Bordetella polysaccharide (Bps), a member of the poly-β-1,6-N-acetyl-D-glucosamine (PNAG/PGA) family of polysaccharides promotes respiratory tract colonization of Bp by resisting killing by antimicrobial peptides (AMPs). Genetic deletion of the bpsA-D locus, as well as treatment with the specific glycoside hydrolase Dispersin B, increased susceptibility to AMP-mediated killing. Bps was found to be both cell surface-associated and released during laboratory growth and mouse infections. Addition of bacterial supernatants containing Bps and purified Bps increased B. pertussis resistance to AMPs. By utilizing ELISA, immunoblot and flow cytometry assays, we show that Bps functions as a dual surface shield and decoy. Co-inoculation of C57BL/6J mice with a Bps-proficient strain enhanced respiratory tract survival of the Bps-deficient strain. In combination, the presented results highlight the critical role of Bps as a central driver of B. pertussis pathogenesis. Heterologous production of Bps in a non-pathogenic E. coli K12 strain increased AMP resistance in vitro, and augmented bacterial survival and pathology in the mouse respiratory tract. These studies can serve as a foundation for other PNAG/PGA polysaccharides and for the development of an effective Bp vaccine that includes Bps.

Traditionally, whooping cough or pertussis caused by the obligate human pathogen Bordetella pertussis (Bp) is described as an acute disease with severe symptoms. However, many individuals who contract pertussis are either asymptomatic or show very mild symptoms and yet can serve as carriers and sources of bacterial transmission. Biofilms are an important survival mechanism for bacteria in human infections and disease. However, bacterial determinants that drive biofilm formation in humans are ill-defined. In the current study, we show that Bp infection of well-differentiated primary human bronchial epithelial cells leads to formation of bacterial aggregates, clusters, and highly structured biofilms which are colocalized with cilia. These findings mimic observations from pathological analyses of tissues from pertussis patients. Distinct arrangements (mono-, bi-, and tri-partite) of the polysaccharide Bps, extracellular DNA, and bacterial cells were visualized, suggesting complex heterogeneity in bacteria-matrix interactions. Analyses of mutant biofilms revealed positive roles in matrix production, cell cluster formation, and biofilm maturity for three critical Bp virulence factors: Bps, filamentous hemagglutinin, and adenylate cyclase toxin. Adherence assays identified Bps as a new Bp adhesin for primary human airway cells. Taken together, our results demonstrate the multi-factorial nature of the biofilm extracellular matrix and biofilm development process under conditions mimicking the human respiratory tract and highlight the importance of model systems resembling the natural host environment to investigate pathogenesis and potential therapeutic strategies.

We determined the molecular epidemiology of Bordetella pertussis isolates to evaluate its potential impact on pertussis reemergence in a population of Mexico. Symptomatic and asymptomatic cases were included. Pertussis infection was confirmed by culture and real-time polymerase chain reaction (PCR). Selected B. pertussis isolates were further analysed; i.e. clonality was analysed by pulsed-field gel electrophoresis (PFGE) and ptxP-ptxA, prn, fim2 and fim3 typing was performed by PCR and sequencing. Out of 11 864 analysed samples, 687 (5.8%) were positive for pertussis, with 244 (36%) confirmed by both culture and PCR whereas 115 (17%) were positive only by culture and 328 (48%) were positive only by PCR. One predominant clone (clone A, n = 62/113; 55%) and three major subtypes (A1, A2 and A3) were identified by PFGE. All 113 selected isolates had the allelic combination ptxP3-ptxA1. The predominant clone A and the three major subtypes (A1, A2 and A3) corresponded to the emerging genotypes ptxP3-ptxA1-prn2-fim2-1-fim3-2 and ptxP3-ptxA1-prn2-fim2-1-fim3-1. In conclusion, the presence of an endemic clone and three predominant subtypes belonging to the genotypes ptxP3-ptxA1-prn2-fim2-1-fim3-2 and ptxP3-ptxA1-prn2-fim2-1-fim3-1 were detected. This finding supports the global spread/expansion reported for these outbreaks associated genotypes.

Objective. To determine the rate of Bordetella pertussis infection in children admitted to a paediatric emergency hospital service.Material and methods. The positivity rate to B. pertussis infection was determined in 344 enrolled children under five-year old measured by culture, RT-PCR, clinical and laboratory predictors.Results. The positive rate of B. pertussis infection was 5.40% (95%CI=2.24-13.05) by culture, 16.12% (95%CI=2.42-29.84) by RT-PCR, and 36.66% (95%CI=18.36-54.97) by the laboratory predictor.Conclusions. Clinical and laboratory predictors represent a suitable tool to improve the diagnosis and prevention of pertussis by health services in the region of study.

Infections and disease caused by the obligate human pathogen Bordetella pertussis (Bp) are increasing, despite widespread vaccinations. The current acellular pertussis vaccines remain ineffective against nasopharyngeal colonization, carriage, and transmission. In this work, we tested the hypothesis that Bordetella polysaccharide (Bps), a member of the poly-β-1,6-A-acetyl-D-glucosamine (PNAG/PGA) family of polysaccharides promotes respiratory tract colonization of Bp by resisting killing by antimicrobial peptides (AMPs). Genetic deletion of the bpsA-D locus, as well as treatment with the specific glycoside hydrolase Dispersin B, increased susceptibility to AMP-mediated killing. Bps was found to be both cell surface-associated and secreted during laboratory growth and mouse infections. Addition of bacterial supernatants containing Bps and purified Bps increased B. pertussis resistance to AMPs. By utilizing ELISA, immunoblot and flow cytometry assays, we show that Bps functions as a dual surface shield and decoy by inhibiting AMP binding. Co-inoculation of C57BL/6J mice with a Bps-proficient strain enhanced respiratory tract survival of the Bps-deficient strain. In combination, the presented results highlight the critical role of Bps as a central driver of B. pertussis pathogenesis. Heterologous production of Bps in a non-pathogenic E. coli K12 strain increased AMP resistance in vitro, and augmented bacterial survival and pathology in the mouse respiratory tract. Therefore, by conferring virulence traits across bacterial genera, Bps transforms a primarily intestinal and urinary tract bacterium into a respiratory pathogen. These studies can serve as a foundation for other PNAG/PGA polysaccharides and for the development of an effective Bp vaccine that includes Bps.Author summaryPertussis or whooping cough, caused by the obligate human pathogen Bordetella pertussis (Bp), is resurging in many countries. Currently, the mechanism by which B. pertussis subverts and resists host immunity is poorly known. In this manuscript, we examined the role of the B. pertussis polysaccharide Bps in promoting resistance to antimicrobial peptides (AMPs), a critical component of host immune defense. We show that the presence of Bps on the bacterial cell surface enhanced AMP resistance. Bps was secreted both during bacterial growth and during mouse infections. We further found that Bps functioned both as a surface shield and decoy, thereby inhibiting AMP binding. Simultaneous infection of mice with Bps-proficient and Bps- deficient strains resulted in greater survival of the Bps-deficient strain in the mouse respiratory tract. Finally, production of Bps in a non-pathogenic E. coli strain increased AMP resistance in vitro, and increased bacterial survival and heightened pathology in the mouse respiratory tract. Our study provides new insights into how B. pertussis has evolved to survive in the mammalian respiratory tract. Additionally, these studies underscore the potential of a single virulence factor to convert a non-pathogenic bacterium into a respiratory tract pathogen.

Here, we report the draft genome sequences of 4 Bordetella pertussis isolates which correspond to major clones isolated between 2008 and 2014 from two outbreaks in northeastern Mexico. The B. pertussis clinical isolates belong to the ptxP3 lineage, and they are grouped into two major clusters, defined by the fimH allele.